Systemic therapies are effective initially in controlling and reversing tumor growth. However, residual cancers will invariably re-grow despite this initial response. Historically, investigation of treatment resistance has focused on the identification of acquired genetic alterations in bulk tumor epithelial cells that confer resistance to specific agents, or to multiple agents. But our recent data supports the existence of a small but distinct subpopulation of cancer cells present in the original tumor that are greatly enriched in residual cancers after conventional therapies. These residual cancer cells are characterized by their relative quiescence and resistance to therapy, yet they possess enhanced self-renewal capacity like that of stem cells. We therefore hypothesize that a unique subpopulation of cancer cells present in the original tumors are intrinsically resistant to conventional therapies, and are responsible for tumor initiation and cancer regrowth. To test our hypothesis, we propose: (1) To determine whether treatment-resistant breast cancers from our neoadjuvant trials are enriched for subpopulations of cells with self-renewal and tumor-initiating capacity. We will use cell surface markers and fluorescent vital dye retention to identify these cells and purify them by FACS. Self-renewal and tumorigenicity will be assessed by in vitro mammosphere (MS) assays and xenograft transplantation, respectively. (2) To define the regulatory genes and signaling pathways responsible for treatment resistance and self-renewal in human breast cancers after conventional chemotherapy. This will be done using mRNA and BAG methylation microarrays to determine gene expression and epigenetic similarities among label-retaining MS-initiating cells, CD44+/CD24- putative breast cancer stem cells, and chemoresistant residual cancer cells, and differences from differentiated primary invasive breast cancer cells. (3) To determine whether suppression of self-renewal and treatment resistance pathways can improve existing cancer therapies using MS-formation assays as well as our novel primary xenograft mouse models in animal clinical trials, leading to human trials targeting this tumor re-initiating subpopulation directly. If our hypothesis is correct, then relapse after apparently successful therapy is due to the persistence of these resting, therapy-resistant tumor cells, thereby allowing tumor re-initiation. If so, approaches for anticancer drug development must be changed fundamentally to target these rare tumor-initiating cells, rather than only cells of the bulk tumor as has been done to date.